In the field of clinical examinations, calcium ions in body fluids such as blood and urine are quantitatively analyzed for diagnosis of endocrine disorders such as parathyroid abnormality, circulatory diseases such as hypertension and arteriosclerosis, and the like. Quantitative analysis of calcium ions is roughly divided into two methods: one is an instrumental analysis exemplified by atomic-absorption spectroscopy and electrode method; and the other is chelate color formation exemplified by orthocresolphthalein complexone (hereinafter abbreviated as OCPC) method. In recent years, enzyme methods for calcium analysis have been proposed, in which activation or inhibition of an enzyme by calcium is utilized. For example, JP-A-62-195297 (the term "JP-A" as used herein means an "unexamined published Japanese patent application") discloses an analytical composition which utilizes the activation of phospholipase D by calcium ions; JP-A-62-36199 discloses an analytical composition which utilizes activation of calmodulin by calcium and activation of a calmodulin-dependent enzyme by the thus activated calmodulin; and JP-A-1-231896 discloses an analytical composition which utilizes activation of phospholipase A.sub.2 by calcium ions. JP-A-2-142498 proposes an analytical composition which utilizes inhibition of pyruvate kinase by calcium ions. JP-A-2-276597 proposes a determination method using activation of amylase calcium ions.
The above-described instrumental analyses give relatively accurate values but require expensive instruments and complicated operations. In particular, atomic-absorption spectroscopy requires dilution of a sample under analysis, and the dilution operation causes errors in measurement.
Chelate color formation typically including OCPC is widely used for reasons that measurements can be made with a simple calorimeter, requiring neither special analytical instruments nor dilution operation. However, the results obtained include considerable factors of error because, for one thing, the degree of color formation is dependent on temperature, pH, etc. and, for another thing, specificity to calcium ions is not sufficient for avoiding influences of other co-present substances.
The up-to-date enzymatic methods have a narrow range of calcium analysis or easily are influenced by substances which are co-present and therefore lack reproducibility. For example, the method of using calmodulin (JP-A-62-36199) is disadvantageous in that calmodulin is hardly available, the measurable range of calcium concentration is narrow so that dilution of a sample is needed, and the two-stage activation system requires a long reaction time. The method of using phospholipase A.sub.2 (JP-A-1-231896) and the method of using pyruvate kinase (JP-A-2-142498) cause errors when applied to assay of serum because these enzymes are present in blood. Besides, the method of using pyruvate kinase has poor accuracy since it utilizes enzyme inhibition by calcium ions. Further, the method of using amylase (JP-A-2-276597) has a problem in reproducibility of determined value since blank reaction is too large. The method of using phospholipase D (JP-A-62-195297) accomplishes measurements with accuracy because the enzyme used is not present in blood, but still is influenced by co-present salts and, in addition, is unsuitable for continuous measurements.